The goal of this research proposal is to understand the structure and mechanism of dopamine beta-hydroxylase (DBH) and peptidyl alpha-amidating monooxygenase (PAM). The former enzyme, DBH, is responsible for the formation of norepinephrine, a neurotransmitter, from dopamine. The latter enzyme, PAM, is responsible for the production of bioactive peptide hormones. Both are classified as Cu(II)-activated monooxygenases and they use O2 and ascorbate as cosubstrates. The specific aims with DBH are as follows: 1) to determine the nature of the "activated" oxygen species and copper centers in the chemical mechanism using alternate substrates, mechanism-based inhibitors and spectroscopic methods (EPR, spin-echo EPR, EXAFS). 2) to isolate and determine the structure of the enzyme adducts (peptides) formed upon inactivation of DBH by several classes of mechanism-based inhibitors. 3) to determine the nature of the "transition-state" in the C-H bond activation step by determining the intrinsic isotope effect for a series of ring- substituted phenethylamine substrates. 4) to clone DBH, determine the primary amino acid sequence of the enzyme from the DNA sequence and express the active enzyme. The specific aims with PAM as follows: 1) to establish the role of O2 in the initial oxidation reaction. Isotopically labeled (180)O2 and (180)O2 and (18o)H2O will be used to determine if the terminal gly residue of peptides is hydroxylated in the initial step or whether the gly undergoes dehydrogenation (-2H.) by a cofactor followed by hydrolysis of an imine intermediate. 2) to establish stereochemistry of the hydrogen abstraction step by using chirally labeled (R)-3H and (s)-3H gly terminated peptides. 3) to synthesize several alpha-substituted (terminal) gly containing peptides as potential mechanism-based inhibitors with the intention of a) determining mechanistic steps in the reaction and b) isolating and identifying active site amino acid residues. 4) to improve the purification of the enzyme by isolating monoclonal antibodies to PAM for the preparation of antibody-linked Sepharose columns. 5) to establish the stoichiometry of peptide substrate, O2, and ascorbate in the formation of alpha-amide containing peptide and glyoxylate. The role of copper in the reaction will be studied by EPR methods.